Machine learning on alignment features for parent-of-origin classification of simulated hybrid RNA-seq.

BACKGROUND: Parent-of-origin allele-specific gene expression (ASE) can be detected in interspecies hybrids by virtue of RNA sequence variants between the parental haplotypes. ASE is detectable by differential expression analysis (DEA) applied to the counts of RNA-seq read pairs aligned to parental references, but aligners do not always choose the correct parental reference. RESULTS: We used public data for species that are known to hybridize. We measured our ability to assign RNA-seq read pairs to their proper transcriptome or genome references. We tested software packages that assign each read pair to a reference position and found that they often favored the incorrect species reference. To address this problem, we introduce a post process that extracts alignment features and trains a random forest classifier to choose the better alignment. On each simulated hybrid dataset tested, our machine-learning post-processor achieved higher accuracy than the aligner by itself at choosing the correct parent-of-origin per RNA-seq read pair. CONCLUSIONS: For the parent-of-origin classification of RNA-seq, machine learning can improve the accuracy of alignment-based methods. This approach could be useful for enhancing ASE detection in interspecies hybrids, though RNA-seq from real hybrids may present challenges not captured by our simulations. We believe this is the first application of machine learning to this problem domain.

Structural evidence for MADS-box type I family expansion seen in new assemblies of Arabidopsis arenosa and A. lyrata.

Arabidopsis thaliana diverged from A. arenosa and A. lyrata at least 6 million years ago. The three species differ by genome-wide polymorphisms and morphological traits. The species are to a high degree reproductively isolated, but hybridization barriers are incomplete. A special type of hybridization barrier is based on the triploid endosperm of the seed, where embryo lethality is caused by endosperm failure to support the developing embryo. The MADS-box type I family of transcription factors is specifically expressed in the endosperm and has been proposed to play a role in endosperm-based hybridization barriers. The gene family is well known for its high evolutionary duplication rate, as well as being regulated by genomic imprinting. Here we address MADS-box type I gene family evolution and the role of type I genes in the context of hybridization. Using two de-novo assembled and annotated chromosome-level genomes of A. arenosa and A. lyrata ssp. petraea we analyzed the MADS-box type I gene family in Arabidopsis to predict orthologs, copy number, and structural genomic variation related to the type I loci. Our findings were compared to gene expression profiles sampled before and after the transition to endosperm cellularization in order to investigate the involvement of MADS-box type I loci in endosperm-based hybridization barriers. We observed substantial differences in type-I expression in the endosperm of A. arenosa and A. lyrata ssp. petraea, suggesting a genetic cause for the endosperm-based hybridization barrier between A. arenosa and A. lyrata ssp. petraea.

Spatial and temporal regulation of parent-of-origin allelic expression in the endosperm.

Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either Pacific Biosciences (PacBio) or Oxford Nanopore technologies and achieves a contig NG50 of >21 Mbp on both human and Drosophila melanogaster PacBio data sets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

Regulation of Parent-of-Origin Allelic Expression in the Endosperm.

Genomic imprinting is an epigenetic phenomenon established in the gametes prior to fertilization that causes differential expression of parental alleles, mainly in the endosperm of flowering plants. The overlap between previously identified panels of imprinted genes is limited. To investigate imprinting, we used high-resolution sequencing data acquired with sequence-capture technology. We present a bioinformatics pipeline to assay parent-of-origin allele-specific expression and report more than 300 loci with parental expression bias in Arabidopsis (Arabidopsis thaliana). In most cases, the level of expression from maternal and paternal alleles was not binary, instead supporting a differential dosage hypothesis for the evolution of imprinting in plants. To address imprinting regulation, we systematically employed mutations in regulative epigenetic pathways suggested to be major players in the process. We established the mechanistic mode of imprinting for more than 50 loci regulated by DNA methylation and Polycomb-dependent histone methylation. However, the imprinting patterns of most genes were not affected by these mechanisms. To this end, we also demonstrated that the RNA-directed DNA methylation pathway alone does not substantially influence imprinting patterns, suggesting that more complex epigenetic pathways regulate most of the identified imprinted genes.

The bonobo genome compared with the chimpanzee and human genomes.

Two African apes are the closest living relatives of humans: the chimpanzee (Pan troglodytes) and the bonobo (Pan paniscus). Although they are similar in many respects, bonobos and chimpanzees differ strikingly in key social and sexual behaviours, and for some of these traits they show more similarity with humans than with each other. Here we report the sequencing and assembly of the bonobo genome to study its evolutionary relationship with the chimpanzee and human genomes. We find that more than three per cent of the human genome is more closely related to either the bonobo or the chimpanzee genome than these are to each other. These regions allow various aspects of the ancestry of the two ape species to be reconstructed. In addition, many of the regions that overlap genes may eventually help us understand the genetic basis of phenotypes that humans share with one of the two apes to the exclusion of the other.

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

BACKGROUND: Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. RESULTS: Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. CONCLUSIONS: Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

An improved genome assembly uncovers prolific tandem repeats in Atlantic cod.

BACKGROUND: The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies. RESULTS: By combining data from Illumina, 454 and the longer PacBio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the Atlantic cod genome assembly. The sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. Compared to other vertebrates, the assembly contains an unusual high density of tandem repeats (TRs). Indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these TRs. We show that 21% of the TRs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual. CONCLUSIONS: The inclusion of PacBio reads combined with the use of multiple assembly programs drastically improved the Atlantic cod genome assembly by successfully resolving long TRs. The high frequency of heterozygous TRs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in Atlantic cod populations, which is likely of evolutionary importance.

Exploring structural variation and gene family architecture with De Novo assemblies of 15 Medicago genomes.

BACKGROUND: Previous studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome. RESULTS: Genome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable - estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation. CONCLUSIONS: Analysis of multiple M. truncatula genomes illustrates the value of de novo assemblies to discover and describe structural variation, something that is often under-estimated when using read-mapping approaches. Comparisons among the de novo assemblies also indicate that different large gene families differ in the architecture of their structural variation.

Hybrid assembly with long and short reads improves discovery of gene family expansions.

BACKGROUND: Long-read and short-read sequencing technologies offer competing advantages for eukaryotic genome sequencing projects. Combinations of both may be appropriate for surveys of within-species genomic variation. METHODS: We developed a hybrid assembly pipeline called "Alpaca" that can operate on 20X long-read coverage plus about 50X short-insert and 50X long-insert short-read coverage. To preclude collapse of tandem repeats, Alpaca relies on base-call-corrected long reads for contig formation. RESULTS: Compared to two other assembly protocols, Alpaca demonstrated the most reference agreement and repeat capture on the rice genome. On three accessions of the model legume Medicago truncatula, Alpaca generated the most agreement to a conspecific reference and predicted tandemly repeated genes absent from the other assemblies. CONCLUSION: Our results suggest Alpaca is a useful tool for investigating structural and copy number variation within de novo assemblies of sampled populations.

ThaleMine: A Warehouse for Arabidopsis Data Integration and Discovery.

ThaleMine (https://apps.araport.org/thalemine/) is a comprehensive data warehouse that integrates a wide array of genomic information of the model plant Arabidopsis thaliana. The data collection currently includes the latest structural and functional annotation from the Araport11 update, the Col-0 genome sequence, RNA-seq and array expression, co-expression, protein interactions, homologs, pathways, publications, alleles, germplasm and phenotypes. The data are collected from a wide variety of public resources. Users can browse gene-specific data through Gene Report pages, identify and create gene lists based on experiments or indexed keywords, and run GO enrichment analysis to investigate the biological significance of selected gene sets. Developed by the Arabidopsis Information Portal project (Araport, https://www.araport.org/), ThaleMine uses the InterMine software framework, which builds well-structured data, and provides powerful data query and analysis functionality. The warehoused data can be accessed by users via graphical interfaces, as well as programmatically via web-services. Here we describe recent developments in ThaleMine including new features and extensions, and discuss future improvements. InterMine has been broadly adopted by the model organism research community including nematode, rat, mouse, zebrafish, budding yeast, the modENCODE project, as well as being used for human data. ThaleMine is the first InterMine developed for a plant model. As additional new plant InterMines are developed by the legume and other plant research communities, the potential of cross-organism integrative data analysis will be further enabled.

Araport: the Arabidopsis information portal.

The Arabidopsis Information Portal (https://www.araport.org) is a new online resource for plant biology research. It houses the Arabidopsis thaliana genome sequence and associated annotation. It was conceived as a framework that allows the research community to develop and release 'modules' that integrate, analyze and visualize Arabidopsis data that may reside at remote sites. The current implementation provides an indexed database of core genomic information. These data are made available through feature-rich web applications that provide search, data mining, and genome browser functionality, and also by bulk download and web services. Araport uses software from the InterMine and JBrowse projects to expose curated data from TAIR, GO, BAR, EBI, UniProt, PubMed and EPIC CoGe. The site also hosts 'science apps,' developed as prototypes for community modules that use dynamic web pages to present data obtained on-demand from third-party servers via RESTful web services. Designed for sustainability, the Arabidopsis Information Portal strategy exploits existing scientific computing infrastructure, adopts a practical mixture of data integration technologies and encourages collaborative enhancement of the resource by its user community.

Diagnostic Value of Early Magnetic Resonance Imaging After Acute Lateral Ankle Injury.

We report a retrospective study of 171 consecutive patients with a lateral ankle sprain. All the patients with direct or blunt force trauma were excluded. Within 21 days of injury, 115 (67.25%) patients had undergone magnetic resonance imaging to evaluate for more serious or significant injuries. The average patient age was 44.09 years. Of the 115 patients, 75 (65.23%) had findings noted to be "significant." MRI can serve as a valuable and underused tool in the evaluation of acute lateral ankle injuries. The underuse of MRI might explain the high degree of variability in patients recovering from a lateral ankle sprain.

Association of Vitamin D With Stress Fractures: A Retrospective Cohort Study.

Vitamin D is an essential, fat-soluble nutrient that is a key modulator of bone health. Despite the gaining popularity throughout published medical studies, no consensus has been reached regarding a serum vitamin D level that will guarantee adequate skeletal health in a patient with an increased functional demand. The purpose of the present investigation was to examine the serum concentrations of vitamin D in patients with confirmed stress fractures. A total of 124 patients were included in our retrospective cohort study. Of the 124 patients, 53 had vitamin D levels measured within 3 months of diagnosis. An association was seen in patients with a stress fracture and vitamin D level measured, as 44 (83.02%) of the 53 patients had a serum 25-hydroxyvitamin D level <40 ng/mL. Although an association was seen at our institution in patients with stress fractures and a serum vitamin D concentration <40 ng/mL, a larger and prospective investigation is warranted to further understand the effect of vitamin D level and stress fracture prevention in an active, nonmilitary population.

Lyme Disease Manifestations in the Foot and Ankle: A Retrospective Case Series.

Lyme disease is the result of Borrelia burgdorferi bacterial infection after exposure from a tick bite. A pathognomonic finding in early-stage Lyme disease is an expanding, red macular ring known as erythema migrans. Lyme arthritis is a late-stage manifestation of this disease, affecting the large, weightbearing joints with intermittent pain and swelling. The existing data on Lyme disease and subsequent arthritis have reported manifestations in the lower extremity, primarily in the knee and ankle and less commonly the small joints of the foot. We present a retrospective case series of 11 cases of painful arthritis in the foot and ankle with confirmatory Lyme disease testing.

Evaluation of machine learning models that predict lncRNA subcellular localization.

The lncATLAS database quantifies the relative cytoplasmic versus nuclear abundance of long non-coding RNAs (lncRNAs) observed in 15 human cell lines. The literature describes several machine learning models trained and evaluated on these and similar datasets. These reports showed moderate performance, e.g. 72-74% accuracy, on test subsets of the data withheld from training. In all these reports, the datasets were filtered to include genes with extreme values while excluding genes with values in the middle range and the filters were applied prior to partitioning the data into training and testing subsets. Using several models and lncATLAS data, we show that this 'middle exclusion' protocol boosts performance metrics without boosting model performance on unfiltered test data. We show that various models achieve only about 60% accuracy when evaluated on unfiltered lncRNA data. We suggest that the problem of predicting lncRNA subcellular localization from nucleotide sequences is more challenging than currently perceived. We provide a basic model and evaluation procedure as a benchmark for future studies of this problem.

Multi-symptom Relief with Propylene Glycol-Hydroxypropyl-Guar Nanoemulsion Lubricant Eye Drops in Subjects with Dry Eye Disease: A Post-Marketing Prospective Study.

INTRODUCTION: The study aimed to evaluate multi-symptom relief of dry eye manifestations with the use of propylene glycol-hydroxypropyl-guar (PG-HPG) nanoemulsion lubricant eye drops, among subjects with dry eye disease (DED). METHODS: This was a post-marketing, prospective, single-arm study conducted in the USA. Subjects aged ≥ 18 years, with tear breakup time (TBUT) ≤ 10 s for both eyes, dry eye questionnaire-5 (DEQ-5) "watery eyes" symptom score 1-4, symptoms of burning/stinging, sore and tired eyes as determined by impact of dry eye on everyday living-symptom bother (IDEEL-SB) questionnaire, and IDEEL-SB score 16-65 were included. Subjects were required to complete IDEEL-SB and DEQ-5 at days 0, 14 ± 2, and 28 ± 2, and self-administer one drop of PG-HPG four times daily for 28 ± 2 days. Primary endpoints were change from baseline at day 28 in symptoms of sore, stinging/burning, and tired eyes on IDEEL-SB; and symptom of watery eyes on DEQ-5. Other endpoints evaluated were corneal staining and TBUT at baseline and day 28 ± 2; symptom relief (5-point Likert scale) at day 28 ± 2, and safety. RESULTS: Of 119 subjects enrolled, 95 completed the study (mean ± SD age 61.2 ± 13.0 years; female 69.5%). Mean IDEEL-SB scores reduced significantly from baseline at day 28 for symptoms of aching/sore eyes (change from baseline - 1.0 ± 1.1), burning/stinging eyes (change from baseline - 1.1 ± 0.9), and tired eyes (change from baseline - 1.1 ± 1.0) (all p < 0.0001). Mean DEQ-5 score for watery eye symptoms significantly reduced from baseline at day 28 (change from baseline - 0.9 ± 1.0, p < 0.0001). Corneal staining at day 28 was comparable to baseline. TBUT improved from baseline to day 28. On a Likert scale, more than 50% of subjects reported relief from symptoms of sore, stinging, and burning eyes. Three (3.1%) subjects reported treatment-emergent adverse events (non-ocular). CONCLUSIONS: PG-HPG nanoemulsion lubricant eye drops significantly improved multiple dry eye symptoms in subjects with DED over 28 days, with no new safety concerns. TRIAL REGISTRATION: ClinicalTrials.gov Identifier, NCT05056155.

Approach to the Ankle in Adult Acquired Flatfoot Deformity.

Adult acquired flatfoot is a progressive deformity of the foot and ankle, which frequently becomes increasingly symptomatic. The posterior tibial tendon is most commonly associated with the deformity. A targeted physical examination with plain film radiographs is the recommended initial assessment, which will further guide a physician toward procuring more advanced imaging or toward surgical intervention. In this chapter the authors review the current literature of their approach to the treatment of the ankle in end stage of adult acquired flatfoot deformity.

Assembly algorithms for next-generation sequencing data.

The emergence of next-generation sequencing platforms led to resurgence of research in whole-genome shotgun assembly algorithms and software. DNA sequencing data from the Roche 454, Illumina/Solexa, and ABI SOLiD platforms typically present shorter read lengths, higher coverage, and different error profiles compared with Sanger sequencing data. Since 2005, several assembly software packages have been created or revised specifically for de novo assembly of next-generation sequencing data. This review summarizes and compares the published descriptions of packages named SSAKE, SHARCGS, VCAKE, Newbler, Celera Assembler, Euler, Velvet, ABySS, AllPaths, and SOAPdenovo. More generally, it compares the two standard methods known as the de Bruijn graph approach and the overlap/layout/consensus approach to assembly.